Candida albicans (CA) and Streptococcus mutans (SM) biofilm, as well as antifungal and antimicrobial activity. EG-50 and EG-100 exhibited more irregular surfaces and statistically higher mean (Ra) and root-mean-square (rms) roughness  compared to the CG  (p < 0.05). The biomass and extracellular matrix production by CA and SM and the metabolic activity of SM were significantly decreased in EG-50 and EG-100 compared to CG (p < 0.05). CA and SM growth was inhibited by the pure unpolymerized VP resin (48 h). EG-50 and EG-100 recorded a greater irregularity, higher surface roughness, and decreased CA and SM biofilm formation over the CG.The wide application of additive manufacturing in dentistry implies the further investigation into oral micro-organism adhesion and biofilm formation on vat-photopolymerization (VP) dental resins. The surface characteristics and microbiological analysis of a VP dental resin, printed at resolutions of 50 μm (EG-50) and 100 μm (EG-100), were evaluated against an auto-polymerizing acrylic resin (CG). Samples were evaluated using a scanning electron microscope, a scanning white-light interferometer, and analyzed for  Streptococcus mutans, a major cariogenic bacterial species -2H-tetrazolium hydroxide . A solution of XTT at a final concentration of 200 µg/mL and 0.04 mM of menadione was added to the wells for 4 h in the absence of light. After this time, the color change was measured using a SpectraMax M3 microplate reader at a wavelength of 492 nm .3 yeasts of C. albicans and 1 × 104 CFU of S. mutans were added. The following controls were performed: (i) pure BHI medium, (ii) pure unpolymerized VP resin, and (iii) BHI medium with micro-organisms. The plates were visually read after incubation at 37 °C for 48 h [The antifungal and antimicrobial activities of unpolymerized VP resin were evaluated at concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%, diluted in BHI medium according to modified versions of the CLSI M27-A3 and CLSI M07-A9 (2012) for yeasts and bacteria, respectively. To each of the wells (96-well plate), 1 × 10for 48 h ,43.The statistical analysis was performed using the Statistical Package for Social Science IBM SPSS software for Windows . The adherence to the normal curve was assessed with the Shapiro–Wilk test. After verifying the normal distribution, the control- and experimental-group comparisons of surface roughness and microbiological data were performed using the one-way analysis of variance (ANOVA) followed by Tukey’s and Dunnett’s multiple-comparisons tests, respectively. In all of the analyses, the significance level was set at 5%.SEM images of the auto-polymerizing acrylic and VP resin samples are illustrated in p < 0.05). The experimental groups, EG-50  and EG-100 , recorded the highest mean roughness and root mean square, compared to the control group . As for R3z, a statistically significant difference was observed only in EG-100 compared to CG  (The surface-roughness data are presented in 0.44 nm) . Images C. albicans and S. mutans that were evaluated in the different study groups are presented in The biomass, metabolic-activity, and extracellular-matrix data of the micro-organisms C. albicans was observed in CG in relation to EG-100 at 72 h (p < 0.05). The CG samples also presented a statistically greater quantification of the biomass of S. mutans compared to the experimental groups (EG-50 and EG-100) at 24 h and 72 h (p < 0.05), and to EG-50 at 48 h (p < 0.05) (A significantly higher quantification of the biomass of  < 0.05) .C. albicans among the study groups. However, a statistically higher metabolic activity of S. mutans was observed in CG compared to EG-50 and EG-100 at 48 h (p < 0.05), and in CG compared to EG-50 at 72 h (p < 0.05) (No significant differences were observed in the metabolic activity of  < 0.05) .C. albicans in relation to EG-50 and EG-100 at 24 h (p < 0.05) and 48 h (p < 0.01). Also, the biofilm formed by S. mutans recorded increased records of extracellular matrix production in CG compared to EG-50 and EG-100 at 24 h (p < 0.001) and 72 h (p ≤ 0.001) (CG presented a higher extracellular matrix production of biofilm formation by ≤ 0.001) .C. albicans and S. mutans growth after a 48 h incubation period at 37 °C.Antifungal and antimicrobial activity testing indicated that, with the exception of pure unpolymerized VP resin (100%), all of the VP-resin dilutions and control systems of BHI medium with micro-organisms presented The wide application of additive-manufacturing resources in dentistry implies the further investigation into the capacity of oral micro-organisms to adhere and form biofilm on vat-photopolymerization (VP)-resin substrates. This in vitro study analyzed the surface characteristics of a VP dental resin for additive manufacturing, printed at resolutions of 50 and 100 μm, in order to determine whether there would be a difference in the surface characteristics and microbiological activity compared to an auto-polymerizing acrylic resin. This investigation directly applies to the manufacture of intraoral devices that usually remain in the oral cavity for a long period of time ,5.The analysis of the images obtained with SEM  and withWhen investigating the surface quality of a material-jetting additive-manufacturing process, Udroiu et al.  observedC. albicans and S. mutans biofilm formation in comparison to CG. These findings may be attributed to the influence of the surface energy and the charge of the photopolymers for additive manufacturing on micro-organism colonization and growth [The literature shows that an increased surface roughness above the Ra threshold of 0.2 μm  facilitad growth ,55. Celld growth , and sind growth  presented growth .S. mutans and C. albicans, which is attributed to the phospholipids and teichoic acids of gram-positive bacteria and the sialic acid of the Candida cell wall [The negative surface charge of ell wall , is attrell wall ,59,60,61C. albicans’ ability to adhere to polymeric surfaces, which are important in the initial adherence to the dental resin, providing means for further attachment and colonization [There is also a strong correlation between attractive hydrophobic and repulsive electrostatic forces with nization ,63. The nization . Secondanization .C. albicans than those printed at 0° and 45°. In addition, the 90° printing orientation was chosen as it allowed for less contact with the supporting pillar of the specimen during the printing process, thereby reducing the interference on the sample’s surface.Another factor that may influence microbial colonization is the orientation of the object at the time of photopolymer printing. In 2020, Shim et al.  demonstrLepidium sativum seed oil. Meanwhile, the selection of antimicrobial components needs to be done carefully, as there is a great similarity between human cells and fungi (both eukaryotic organisms), which hampers the use of a substance that only has selective toxicity for pathogens [The potential of microbiological growth inhibition by the photopolymers should also be highlighted. Many authors have previously discussed the benefits of introducing components with antibacterial potential to additive manufacturing, especially against micro-organisms that have, to some degree, bacterial resistance ,66,67,68athogens .S. mutans and C. albicans in its pure unpolymerized form for a period of 48 h. However, once diluted, even at the lowest concentration of BHI (90% photopolymer and 10% BHI), there was a positive microbiological growth. Therefore, further investigation is necessary to evaluate whether the inhibition of microbiological growth in the pure unpolymerized photopolymer is due to the lack of BHI medium nutrients or to a soluble component with antifungal and antimicrobial properties that loses its potential of action when in contact with the BHI medium.The antifungal and antimicrobial potential of photopolymers would be crucial in dentistry with regards to the manufacturing of several intraoral devices such as prostheses and orthodontic retainers. The photopolymer evaluated in the present study inhibited the growth of C. albicans and S. mutans biofilm formation over the auto-polymerizing acrylic resin.VP dental-resin samples recorded a greater irregularity, higher surface roughness, and decreased Surface characteristics and microbiological colonization of VP resins were not significantly affected by the 3D-printing resolutions of 50 and 100 μm.C. albicans and S. mutans growth only in its pure, unpolymerized form for an incubation period of 48 h.VP dental resin inhibited Within the limitation of this in vitro study, the following conclusions can be drawn:


BRAF p.K601E mutation (nine cases). We determined the overall miRNA expression profile with NanoString technology, and both quantitative reverse transcription–PCR and in situ hybridization were used to confirm selected miRNAs. The miRNA signature was found to consistently differentiate specific histotypes and mutational profiles. In contrast to the BRAF p.K601E mutation and classic PTCs, three miRNAs  were substantially overexpressed in the hobnail variant. The current study found that different miRNA signature profiles were linked to unique histological variants and BRAF mutations in PTC. Further studies focusing on the downstream pathogenetic functions of mRNAs in thyroid neoplasms are warranted.Papillary thyroid carcinoma (PTC) is a miscellaneous disease with a variety of histological variants, each with its own mutational profile, and clinical and prognostic characteristics. Identification of microRNA (miRNA) expression profiles represents an important benchmark for understanding the molecular mechanisms underlying the biological behavior of these unique PTC subtypes in order that they be better characterized. We considered a series of 35 PTC samples with a histological diagnosis of either hobnail (17 cases) or classical variant (nine cases) and with a specific  Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, accounting for 80%–90% of all tumors affecting this gland, and predominantly affects women , 2. DespBRAF p.V600E is the most common mutation in these tumors, which is present in 50%–65% of HoV-PTC cases, followed by TP53 (56%). Recent studies have also reported RET/PTC1 rearrangements and TERT promoter mutations in HoV-PTC . Other  HoV-PTC , 9. BecaBRAF and TERT promoters have been the genes most investigated as possible outcome indicators. BRAF p.V600E mutations are considered reliable preoperative prognostic factors with regard to optimizing the choice of surgical treatment. In fact, this mutation is very frequent in PTC, varying from 14% to 64% of cases , and seems to be a favorable prognostic factor . MiRNAs are small noncoding single-stranded RNA filaments that act as negative regulators of gene expression at the posttranscriptional level. MiRNAs bind the untranslated region (UTR) of target mRNAs and block their translation or degradation, regulating the downstream cascade , 15. TheBRAF p.V600E, with the aim of identifying new markers associated with the characteristic biological behavior of this type of carcinoma.In light of these data, we wanted to undertake a pilot study to test whether there was an association between rare histological variants of PTC with more aggressive clinicopathological features or conversely with more benign course between core genetic mutations of PTC and between miRNA signatures. For these reasons, we decided to trace the miRNA expression profiles of representative samples of p.K601E-PTC, hobnail, and classic variants of PTC, which had been previously investigated for All patients underwent a total thyroidectomy or lobectomy between 2008 and 2016 at the University Hospital of Padua, with a histological diagnosis of PTC. Patient material was collected from the archives of the Surgical Pathology Unit at Padua University Hospital . Hematoxylin and eosin-stained slides from formalin-fixed paraffin-embedded tissues were reviewed by two pathologists (GP and FG).BRAF.All cases of HoV-PTC and p.K601E-PTC were selected. Nine CV-PTC cases were included as a control cohort. In all patients, a fine-needle aspiration biopsy was performed on a suspicious node at each ultrasound examination and part of the biopsied material was used for the molecular evaluation of the mutational status of All patients involved in the study gave their written informed consent, and the study was approved by the Center’s Ethics Committee of the University Hospital of Padua. All experiments were performed in accordance with relevant guidelines and regulations.BRAF gene was extracted from the primary tumor of all study patients. Since each sample was initially fixed in formalin and embedded in paraffin for storage, a deparaffining process was carried out for DNA extraction using a “DNeasy Blood & Tissue Handbook” according to the manufacturer’s instructions. After this procedure, qualitative control of the extracted DNA and the determination of its concentration were performed.The genetic material necessary for the molecular analysis of the Once the DNA was extracted, the specific sequences encoding the gene under study were amplified by PCR. Each amplification reaction was verified by electrophoresis through a 2% (P/V) agarose gel in Tris-acetate-EDTA (50×). Using Millipore Microcon columns , the amplification products were separated from primers, proteins, salts, and resins. Direct sequencing of the aforementioned exons was performed using a Big-dye Terminator protocol according to the manufacturer’s instructions. Subsequently, each product was purified using Autoseq G-50 Dye Terminator Removal Kit  columns and then processed by an automatic sequencer ABI PRISM .Total RNA extraction was performed using an Ambion Recover All Isolation Kit , according to the manufacturer’s instructions. The purity and quality of the extracted RNA were determined by Bio-Analyser .® Human v2 miR Expression Assay Kit  as previously described . Data were extracted using an nCounter® RCC Collector (NanoString). Six samples (three HoV-PTC and three p.K601E-PTC) failed quality control and were excluded from further analysis. Raw data, which were proportional to copy number, were log-transformed and normalized by the quantile method after application of a manufacturer-supplied correction factor for several miRNAs  for visual inspection. Only miRNAs with a P-value <0.01 were included in heatmaps. The color red indicates the strong expression of a miRNA, whereas a green point mirrors a reduced level of a determined miRNA.This assay detects 800 endogenous miRNAs, five housekeeping transcripts plus six positive and six negative controls. About 150 ng of each total RNA sample was used as input into the nCounter Human miRNA sample preparation. Hybridization was conducted for 16 h at 65°C. Subsequently, the strip tubes were placed into the nCounter Prep Station for automated sample purification and subsequent reporter capture. Each sample was scanned for 555 fields of view on an nCounterl miRNAs . Data wel miRNAs , 20. P-vg method . Raw dat® Analysis  was investigated by quantitative reverse transcription (qRT)–PCR.Expression of three of the most important dysregulated miRNAs detected using NanoString nCounterFor qRT–PCR analysis, an NCodeTM miRNA qRT–PCR kit was used  according to the manufacturer’s instructions with the following primers:- hsa-miR-21-5p (primer: 5′-cgg tag ctt atc aga ctg atg ttg a-3′)- hsa-miR-146b-5p (primer: 5′-ggg tga gaa ctg aat tcc a-3′)- hsa-miR-205-5p (primer: 5′-ggt cct tca ttc cac cg-3′)RNU6B nuclear RNA expression (5′-gtt ggc tct ggt gca gg gcc tcc gag gta ttc gca-3′). Reactions were performed in a LightCycler 480 Real Time PCR cycler System . All qRT–PCR reactions were conducted in duplicate, and the results were calculated according to the comparative CT method. This method uses the equation 2-dCt, where Ct is the threshold cycle , and dCt is the difference between the mean Ct of the gene sample and the mean Ct of the RNU6B probe.Results were normalized relative to the values of In situ hybridization (ISH) was conducted on tissues of all samples under examination. The ISH was performed using a signal amplification system  following the protocol provided by the manufacturer and modifications made by Yamamichi et al.  was used at a final concentration of 200 nM. The slides were then washed in TBST and then GenPointTM stringent wash solution (at 54°C for 30 min). The slides were exposed to a fixative containing H2O2 (DakoCytomation) for 20 min and then a further solution fixative (DakoCytomation) for 30 min. The slides were then exposed to primary streptavidin-horseradish peroxidase antibody, a biotinylated tiamide, a streptavidin-labeled secondary antibody, and a solution chromogen nitroblue tetrazolium, according to the manufacturer’s instructions. The sections were then mildly counterstained with hematoxylin and washed with TBST and water before assembly.The sections on slides were incubated at 60°C for 30 min, deparaffinized, and subsequently treated with proteinase K (DakoCytomation) for 30 min at room temperature. After gentle rinsing in distilled water and immersion in a 95% ethanol solution for 10 s, sections were air-dried. They were then prehybridized at 49°C–56°C for 1 h with RNA ISH buffer . They were subsequently incubated overnight at 49°C–56°C in RNA ISH buffer containing miRCURYTM LNA probes and labeled with biotin for the detection of hsa-miR-21 . As a control, a probe . A P < 0.05 was considered statistically significant.The BRAF p.K601E FV-PTC). Clinicopathological features are summarized in Of the original 32 patients recruited with a histological and molecular diagnosis of HoV-PTC and p.K601E-PTC, 26 patients were included in our study  of HoV-PTC cases and in 4/9 (44%) of CV-PTC cases. A p.V600E mutation was not found in any patient with p.K601E, as previously detected in cytological specimens. All BRAF p.K601E mutations were identified in histological samples.A BRAF p.K601E FV-PTC cases, and in a randomly selected cohort of nine CV-PTC cases.In order to define miRNAs that distinguished between patients with characteristic histological variants and mutational profiles, we performed miRNA expression analysis in a cohort of 17 HoV-PTC cases, nine P < 0.05) in a comparison between HoV-PTC and BRAF p.K601E FV-PTC cases . If we compare only the HoV-PTC p.V600E mutated subgroup with BRAF p.K601E FV-PTC cases, a large set of 324 miRNAs were significantly deregulated.Dividing the cohort of HoV-PTC cases based on the results of P < 0.05). P-value <0.01.When HoV-PTC and CV-PTC samples were compared, 40 miRNAs were found to be downregulated in HoV-PTC patients, while 14 were overexpressed . Instead, comparing p.V600E CV-PTC to HoV-PTC wild type (wt), 43 miRNAs showed statistically significant differences in expression levels.When the analysis focused on BRAF p.K601E FV-PTC and CV-PTC samples, a set of 153 miRNAs were found to be deregulated (P < 0.05); in particular, only 14 were overexpressed in BRAF p.K601E FV-PTC patients, while the others were downregulated. P-value <0.01.When the analysis compared BRAF p.V600E analysis, we noted that a set of 65 miRNAs was deregulated between the two subgroups. In particular, only three miRNAs were upregulated in a BRAF p.K601E FV-PTC compared to p.V600E CV-PTC cohort, while all the others were downregulated (P < 0.05).Dividing the cohort of CV-PTC cases on the basis of a BRAF p.K601E mutation, miRNAs overexpressed in these samples might be important in determining the biological behavior of disease; (2) overexpressed rather than silenced miRNAs might be easier to detect in tissues and biological fluids. The expression of the three miRNAs  deregulated in HoV-PTC vs. BRAF p.K601E FV-PTC and CV-PTC detected in the cohorts by nCounter® was confirmed in the same cohorts (n = 35) using qRT–PCR. All three miRNAs were shown to be significantly upregulated in HoV-PTC patients  were found to be overexpressed in all BRAF wt samples, while miR-205 was overexpressed in CV-PTC wt vs. CV-PTC p.V600E but downregulated in HoV-PTC wt vs. HoV-PTC p.V600E  since the hobnail variant is associated with a worse prognosis than those of patients with the classic variant and patients Table 5.® and qRT–PCR findings, miR-21 and miR-205 expressions were investigated in all samples (n = 35) by ISH analysis. The expression of miR-21 and miR-205 was identifiable as a granular blue cytoplasmic stain that was scored according to a three-tiered scale . In HoV-PTC, miR-21 was significantly overexpressed: all (9/9) wt HoV-PTC cases were given a score of 2; 63% (5/8) of p.V600E HoV-PTC cases were scored 2, and 37% (3/8) were scored 1 . Instead, all BRAF p.K601E FV-PTC cases were given a score of 0; all (5/5) wt CV-PTC cases and 75% (3/4) of p.V600E CV-PTC cases were given a score of 1, while 25% (1/4) of p.V600E CV-PTC cases were given a score of 2  for the stain. CV-PTC cases were principally given a score of 1 . After a subdivision of the two cohorts (HoV-PTC and CV-PTC) according to molecular results for BRAF p.V600E, a statistically significant difference was found in the expression profile of the same cohorts  and between different cohorts . Among the more deregulated miRNAs, three miRNAs primarily emerged: miR-21-5p, miR 146b-5p, and miR-205-5p. Validation analysis (RT–PCR and ISH) confirmed these results.Our analysis revealed significant differences in terms of a miRNA expression profile between the different cohorts analyzed. In particular, 46 miRNAs were deregulated in a comparison of HoV-PTCs and PDCD4, which negatively regulates protein expression and is a well-known tumor suppressor. The importance of this molecular pathway (which might not necessarily be the sole one) in regulation by miR-21-5p is supported by the finding that miR-21-5p upregulation consistently parallels programmed cell death 4 protein downregulation  affects mir-146 . NuclearIn particular, the overexpression of miR-205-5p in the hobnail variant vs. p.K601E-PTC is noteworthy. Importantly, miR-205 is a driver miRNA whose deregulation may induce cancer development and progression , 37. MorBRAF mutations, which are associated with a different prognosis, are characterized by different miRNA expression profiles. The miRNAs, miR-21-5p, miR-146b-5p, and miR-205-5p, were the most significantly overexpressed miRNAs in the analyzed cohorts. In light of these data, although results are preliminary, these markers appear to play a role in PTC carcinogenesis and, in particular, seem to correlate with its different biological behavior. This is a seminal study that will be deepened considering the most relevant emerged miRNAs in large series for each of the groups. One of the limitations of this study is the low number of analyzed samples mainly due to the rarity of the categories considered such as the p.K601E BRAF mutation and the hobnail variant. Further studies are necessary in order to validate these results in a wider population-based cohort and to understand the exact mechanism of action of these miRNAs.In conclusion, PTC is a heterogeneous neoplasm from both morphological and molecular points of view. The present study highlights this by demonstrating how the different histological variants and specific The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the University Hospital of Padua Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.FG designed the study, performed the research, analyzed the data, and wrote the article. SC, IMB, MF, NV, MR, FV, ADT, CM, and GP designed the study and performed the research. MS, JCH, LB, GM, MG, LC, and SB contributed to the acquisition of clinical samples and data and to the statistical analysis. All authors contributed to the article and approved the submitted version.This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sectors. MF is supported by a grant from the Italian Health Ministry/Veneto region research program NET-2016–02363853 and Fondazione AIRC under 5 per mille, grant ID 22759.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Purpose: The rearranged during transfection (RET) receptor tyrosine kinase plays a key role in transducing signals related to cell growth and differentiation. Ret mutant mice show abnormal retinal activity and abnormal levels and morphology of bipolar cells, yet die on the 21st day after birth as a result of renal underdevelopment. To extend the observation period, we generated the Ret conditional knockout lx/lxChx10-Cre;C-Ret mouse model and analyzed the retinal function and morphological changes in mature and aging lx/lxChx10-Cre;C-Ret mice.Methods: Retina-specific depletion of Ret was achieved using mice with floxed alleles of the Ret gene with CHX10-driven Cre recombinase; floxed mice without Cre expression were used as controls. Retinal function was examined using electroretinography (ERG), and 2-, 4-, 12-, and 24-month-old mice were analyzed by hematoxylin staining and immunohistochemistry to evaluate retinal morphological alterations. The ultrastructure of photoreceptor synapses was evaluated using electron microscopy.Results: The results of the ERG testing showed that b-wave amplitudes were reduced in lx/lxChx10-Cre;C-Ret mice, whereas a-waves were not affected. A histopathological analysis revealed a thinner and disorganized outer plexiform layer at the ages of 12 and 24 months in lx/lxChx10-Cre;C-Ret mice. Moreover, the data provided by immunohistochemistry showed defects in the synapses of photoreceptor cells. This result was confirmed at the ultrastructural level, thus supporting the participation of Ret in the morphological changes of the synaptic ribbon.Conclusion: Our results provide evidence of the role of Ret in maintaining the function of the retina, which was essential for preserving the structure of the synaptic ribbon and supporting the integrity of the outer plexiform layer. Through this special synapse, visual signals are transmitted from photoreceptors to bipolar and horizontal cells . Morpholvesicles . A previvesicles . TherefoTo protect central nervous system (CNS) cells, mutation-independent neuroprotective strategies—such as the glial-cell-line-derived neurotrophic factor (GDNF), the brain-derived neurotrophic factor (BDNF), the nerve growth factor (NGF), and the ciliary neurotrophic factor (CNTF)—have been applied to, and their therapeutic potential has been demonstrated in, the management of retinal impairment in various animal models . GDNF isin vitro (rd1) mouse model , as well as a severely disrupted OPL with very sparse dendrites and axons of horizontal cells  with mice expressing Cre recombinase under the control of the Chx10 gene. The Chx10 gene is specifically expressed in the retinal progenitor cells at the early stage of eye development (E11.5), followed by a restricted expression in the bipolar cells as the progenitor cells differentiate and exit the cell cycle  and/or time-  specific manner when straight knockout of the genes of interest causes embryonic lethality. In this study, we aimed to investigate the function of  unknown . To overll cycle . We analRet < tm1.1Kln >Ret mice were generated by Dr. Klein (Ret gene with Lox P sites flanking exon 12 (lx/lxC-Ret mice hereafter). The generation of conditional knockout mice with Ret gene deletion in the retina was achieved by crossing lx/lxC-Ret mice carrying LoxP sites flanking exon 12 of the Ret gene . All mice were housed in groups of four to five animals per cage in a room that was kept at 23 ± 1°C and 55% ± 5% humidity with a 12-h light/dark cycle, and were given ad libitum access to food and water. All mice used in our experiments were genotyped to confirm the absence of the rd8 and rd1 mutations as they may be present in vendor lines and subsequently confound ocular-induced mutant phenotypes  according to the manufacturer’s instructions. Mice homozygous for lx/lxC-Ret were identified using the C-Ret forward (5′-CCA ACA GTA GCC TCT GTG TAA CCC C-3′) and reverse (5′-GCA GTC TCT CCA TGG ACA TGG TAG CC-3′) primers span the loxP in intron 12 ((Opa1) forward (5′-GAG CTG AGA GGG AGT GAA GAG AGG-3′) and reverse (5′-CCC AAA ACT CCT TTA TCC CAG TGA C-3′) primers could serve as the positive control. Furthermore, the Chx10-Cre mice carried EGFP fused with Cre recombinase; therefore, primers that amplify EGFP were also used to detect the presence of Cre recombinase. The thermal cycling conditions consisted of 30 cycles of 30 s at 94°C, 30 s at 55°C, and 50 s at 72°C. Reactions contained 200 ng of template DNA, 0.5 μM primers, 100 μM dNTPs, 9% glycerol, 2.5 U of Taq polymerase, 1.8 mM MgCl2, and 1× PCR buffer (GIBCO BRL) in a volume of 20 μL. The PCR products were resolved via 2% agarose gel electrophoresis using Gel Red (Invitrogen/Life Technologies) as the visualizing dye. The DNA bands were visualized using a ChemiDoc Imaging System (Bio-Rad).Mice were genotyped and verified using polymerase chain reaction (PCR) analysis. For genotyping, genomic DNA was isolated from a section of mouse tail, optic nerve and retina using an Aquadienntron 12 , top. Op2 in sequence. The intervals between each stimulus varied from 2 to 30 s, and the number of repeats varied from 10 to 4 times. After the completion of dark-adapted recordings, the animals were exposed to a full-field 30 cd/m2 white background for 10 min; subsequent steps were delivered on top of this continuous background. The single-flash stimuli applied after light adaptation consisted of −0.1, 0.1, 0.3, 0.8, 1.0, 1.2, and 1.47 log cd.s/m2. The intervals between each stimulus varied from 1 to 10 s, and the number of repeats varied from 3 to 10 times. A digital band-pass filter ranging from 0.3 to 300 Hz was used to isolate signals after the waves were recorded. The a-wave amplitude was measured from the baseline to the trough of the a-wave, and the b-wave was measured from the trough of the a-wave to the peak of the b-wave.Electroretinography was performed as described previously . After 1Mice were sacrificed at 2, 4, 12, and 24 months of age and their eyes were enucleated and fixed in 4% paraformaldehyde (PFA) in PBS for 1 h at room temperature (RT). For a better retinal infiltration of 4% PFA, corneas were partially removed and then placed in 4% PFA at 4°C for ∼1 h. After washing in PBS three times, the tissues were incubated in a 30% sucrose solution in PBS at 4°C for 3 days. Tissues were embedded in optimum cutting temperature (OCT) compound , snap frozen in liquid nitrogen, and immediately stored at –80°C. Cryosections (14 μm in thickness) were cut and collected on slides . All slides were stored at –80°C before use. Before any staining process, the slides were air dried for 15 min and washed in PBS.Sections were stained with hematoxylin for 1 min, then washed with running water for 5 min. The stained sections were mounted with an aqueous mounting medium  and viewed under a microscope . The brightness and contrast of photomicrographs were adjusted for maximum clarity using Adobe Photoshop CS5 . The retinas of three mice in each group underwent further histological analysis.Sections were blocked with blocking buffer [5% fetal bovine serum (FBS) in PBS containing 0.1% Triton X-100] for 1 h at RT, followed by incubation with the primary antibodies  diluted Retinas were isolated and fixed in 2% glutaraldehyde and 2% PFA in 0.1 M PB at 4°C overnight. After postfixing in 1% osmium tetroxide for 1 h, tissue samples were dehydrated in a graded ethanol series and embedded in epoxy resin . Ultrathin sections (70 nm in thickness) were collected on copper grids and stained with uranyl acetate and lead citrate before examination under a Hitachi H-7100 electron microscope  equipped with a Gatan 832 digital camera .For the quantification of synaptic ribbon conditions, images of the OPL were taken at a magnification of 40,000X. Approximately 500 photoreceptor terminals for each age were examined and classified into different categories of rod-shaped ribbon profiles.2, which was modified from previous studies that chose the area of approximately 200–500 μm away from the optic nerve for quantification (The thickness of the ONL and inner nuclear layer (INL) was quantified from single optical sections. Images were taken at a distance of 200 μm from the optic disc and within fields with a size of 300 × 800 μmfication . Five imt-test. P < 0.05 was considered significant. All analyses were performed using SPSS  and GraphPad Prism 5.0a .All experimental data were assessed by an operator blinded to the genetic condition. The results were presented as the mean ± standard error of the mean (SEM) and statistical significance was determined by independent Student’s lx/lxC-Ret allele in both Chx10-Cre;lx/lxC-Ret and lx/lxC-Ret mice, total DNA from wild-type (WT) and transgenic mice was subjected to PCR analysis with C-Ret primers spanning the intron 12 loxP sequence  was generated by Chx10 BAC transgenes using a “GFP/Cre translational fusion protein” . In photopic serial intensity ERG, there were reductions in amplitude of a- and b- waves in lx/lxChx10-Cre;C-Ret mice, which were statistically significant in some intensities of b-waves. However, there was no obvious difference in the implicit time of a- and b- waves in photopic responses between lx/lxChx10-Cre;C-Ret and lx/lxC-Ret mice. The ERG recordings suggested that the lx/lxChx10-Cre;C-Ret mice may exhibit a greater effect on the function of bipolar cells. Furthermore, these tracings were similar to those of an electronegative ERG corresponding to inner retinal dysfunction.To determine the effect of /lx mice . The C-Ric phase . The imp/lx mice    . Some nu the ONL  and INL  the ONL  using molx/lxC-Ret mice, horizontal cells possessed arborizing processes in the OPL of mice aged 2–24 months , the distribution of which, within cells, is reportedly activity-dependent in the rat model . Thus, v the IPL . A subpo4 months . However4 months . Furtherlx/lxC-Ret mice  . The aforementioned TEM results suggest that the loss of Ret causes a structural defect in the synaptic connection between photoreceptors and bipolar cells. Ultimately, this finding may underlie the abnormal ERG b-wave observed in lx/lxChx10-Cre;C-Ret mice.Furthermore, the appearance of the ribbon synaptic complexes in the 12-month tic site , where gtic site . Howevertoplasm) . Many em/lx mice . In addi/lx mice  and foun/lx mice . FollowiRet-specific knockout mice using the Cre/loxP system and demonstrated a possible role for Ret in retinal function. First, we found severely abnormal ERG patterns, especially those of b-waves, in conditional Ret-knockout retinas. Second, we identified gradually reduced levels of immunoreactivity for α-internexin—a marker of processes of horizontal cells—as lx/lxChx10-Cre;C-Ret mice increased in age. Third, deficiency of Ret in the retina caused the mislocalization of synapses in the ONL, as demonstrated by immunostaining for PKC-α and synaptophysin. Finally, ultrastructural observations of conditional Ret-knockout retinas revealed that the synaptic ribbons were immature and not fully assembled, which may explain the abnormal ERG results.In this study, we generated retinal Ret in the retina was well assessed by full-field ERG in previous studies. Ret hypomorphic mice, which exhibit severely reduced Ret activity, do not survive beyond 3 weeks and display significantly reduced scotopic a-waves, b-waves, and photopic b-waves at postnatal day 18 , or the RET receptor tyrosine kinase can specifically affect distinct photoreceptors and other retinal cell subpopulations.GDNF and other GFLs  share the RET receptor tyrosine kinase as their common signaling receptor. A previous study revealed a thinner and disorganized OPL in ficiency . Moreoveay model . Anotheray model . In our rd1 mouse , and NT-4/5], the GDNF family , and the CNTF, which is a member of the interleukin 6 (IL-6) family of cytokines. Different factors can act in a sequential, simultaneous, additive , or mutual-inhibition fashion. For instance, subpopulations of developing sensory and motor neurons are dependent on the simultaneous action of GDNF and BDNF . MoreoveBassoon (Bsn)-mutant mouse retinas did not attach to the active zone, thus potentially resulting in the failure of synaptic transmission. Moreover, the a-waves of the ERG recordings performed in the Bsn-mutant mouse were not affected, whereas their b-waves, representing the response of the ON bipolar cells, were significantly reduced in amplitude and prolonged in implicit time  and GDNF can promote the long-term survival of target-deprived adult mouse spiral ganglion neurons (Ret, the canonical GDNF receptor, may affect retinal development and cause morphological and physiological alterations in lx/lxChx10-Cre;C-Ret mouse retinas.The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone that is present at conventional synapses. The active zones of synapses are highly organized structures designed for the regulated and site-specific release of neurotransmitters. The function of photoreceptor ribbons was suggested to be the continuous shuttling of vesicles to the active zone, for fusion and the release of glutamate . In a prcit time . Our ult neurons . Further neurons . Therefolx/lxChx10-Cre;C-Ret mice. However, a previous study indicated that the dendrites of rod bipolar cells, normally confined to the OPL, were found to extend into the ONL in normal aging retina and tended to increase in length and incidence with the age (lx/lxC-Ret and lx/lxChx10-Cre;C-Ret mice at the age of 12 months, indicated that the lx/lxChx10-Cre;C-Ret mice may have dysfunctional bipolar cells. Additionally, a previous study speculated that reduced synaptic efficacy may induce new neuronal growth and the formation of ectopic synapses in Basson mutant mice (Our study has documented dendritic sprouting in aging Ret-deficient mouse retinae. The outgrowth of bipolar cell dendrites was reported to be observed under some pathologic conditions, such as retinal detachment , the nob the age . Althougant mice . TherefoRet in retinal development, which is essential to maintain the processes of horizontal cells and preserve the integrity of the OPL by stabilizing the structure of the synaptic ribbons. The lx/lxChx10-Cre;C-Ret mice developed in this study provided a valuable model in which to study Ret function in the retina and enhanced the understanding of Ret function in postnatal development and later stages. Finally, these conditional Ret-knockout mice might be useful for investigating the importance of GFL-mediated RET activation in the retina of animal models of other diseases, such as neurodegenerative diseases or genetic disorders.In conclusion, our results provide evidence of the role of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the National Taiwan University Animal Ethics Committee.W-HP conceived of the study, carried out the ultrastructure observation, drafted the manuscript, and final approval of the manuscript submission. M-LL participated in the design of the experiment and edited the manuscript. W-CH and C-YL carried out the antibody sensitivity test, immunohistochemical staining, and PCR experiment. SL edited the manuscript. Y-JT contributed with the electroretinography and data analysis. L-KY and K-JC contributed with the data analysis and interpretation. C-LC and N-KW designed the study, revised the work critically for important intellectual content, and gave their final approval of the version to be published. All the authors contributed to and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We conducted a systematic review and meta-analysis to assess the available literature regarding the surgical and oncologic outcomes of patients undergoing salvage radical cystectomy (SV-RC) for recurrence or failure of bladder sparing therapy (BST) for muscle-invasive bladder cancer (MIBC).We searched MEDLINE (PubMed), EMBASE and Google Scholar databases in May 2020. We included all studies of patients with ≥ cT2N0/xM0 bladder cancer that were eligible for all treatment modalities at the time of treatment decision who underwent BST including radiotherapy (RTX). A meta-analysis was conducted to calculate the pooled rate of several variables associated with an increased need for SV-RC. Study quality and risk of bias were assessed using MINORS criteria.73 studies comprising 9110 patients were eligible for the meta-analysis. Weighted mean follow-up time was 61.1 months (range 12–144). The pooled rate of non-response to BST and local recurrence after BST, the two primary reasons for SV-RC, was 15.5% and 28.7%, respectively. The pooled rate of SV-RC was 19.2% for studies with a follow-up longer than 5 years. Only three studies provided a thorough report of complication rates after SV-RC. The overall complication rate ranged between 67 and 72% with a 30-day mortality rate of 0–8.8%. The pooled rates of 5 and 10-year disease-free survival after SV-RC were 54.3% and 45.6%, respectively.Approximately one-fifth of patients treated with BST with a curative intent eventually require SV-RC. This procedure carries a proportionally high rate of complications and is usually accompanied by an incontinent urinary diversion.The online version of this article (10.1007/s00345-020-03436-0) contains supplementary material, which is available to authorized users. Data from large meta-analyses suggest that bladder sparing therapy (BST) could offer equal oncologic outcomes compared to radical cystectomy (RC) in well-selected patients with muscle-invasive bladder cancer (MIBC) –3. The mPatients usually would prefer a BST, as it is considered tolerable due to its minimal invasiveness with genuinely manageable toxicity , 13. HowWe, therefore, conducted a systematic review to evaluate the surgical and oncologic outcomes following SV-RC after BST with a curative intent. We also performed a meta-analysis to investigate the pooled incidence rate of SV-RC and assessed the variables associated with an increased likelihood of requirement for SV-RC.salvage cystectomy, rescue surgery, radiotherapy, trimodal therapy, bladder sparing therapy, multimodal therapy, bladder preservation, radiotherapy, radiochemotherapy, pelvic radiation and muscle invasive bladder cancer. Additionally, reference lists of the retrieved articles were analyzed to identify further studies. Studies lacking original patient data, abstracts, and non-English articles were excluded. The primary outcome of interest was the pooled rate of SV-RC, as well as the surgical and oncologic outcomes of patients treated with SV-RC for BST failure. Secondary outcomes of interest were variables associated with a previously reported increased likelihood for SV-RC [This meta-analysis was carried out based on the guidelines of the Preferred Reporting Items for Meta-Analyses of Observational Studies in Epidemiology Statement and registered on PROSPERO (ID: CRD42020187685) . In May or SV-RC .We included all studies of patients with ≥ cT2N0/xM0 bladder cancer who underwent BST which included radiotherapy (RTX). Patients had to be eligible for all treatment modalities at the time of first-line treatment decision. The choice of treatment modality was usually based on patients and/or physicians’ preferences as well as participation in clinical trials. Studies that focused explicitly on elderly patients or patients unfit for surgery (at time of BST) were excluded, as there are limited treatment options for these patients and SV-RC will frequently not be feasible despite being otherwise required. To focus on modern surgical and radiation treatment, we only included studies published between January 2000 and May 2020. To analyze a homogeneous cohort, we excluded studies that focused on primary partial cystectomy or lymph node dissection (LND), brachytherapy or only TURBT/chemotherapy as part of BST. Studies reporting the outcome of SV-RC following pelvic radiotherapy for non-bladder cancer malignancies were excluded. Patient cohorts that focused on pure variants of urothelial carcinoma were also excluded, as there is little data on the value of BST in these patients. Studies reporting results of mixed cohorts were included. Repeated publications from the same authors or institutions were excluded, except for publications with updated or new data from the same institution.Data were extracted independently by two authors. Titles and abstracts were screened to determine whether they met inclusion criteria. Full-text publications of all studies not primarily excluded were obtained and reviewed to check eligibility. If rates were not reported, they were calculated using the number of included patients as denominator, whenever it was possible. Non-response to BST was defined as a stable or progressive disease within 6 months after the initial treatment. Perioperative complications were defined as any complications within 30 days of SV-RC. Early SV-RC was defined as a SV-RC within 6 months of BST. Local recurrence was defined as intravesical recurrence. Missing data were obtained by contacting the authors of the relevant studies, if needed. All discrepancies regarding data extraction were resolved by consensus with co-investigators.As the vast majority of included studies were non-randomized interventional studies, assessment of study quality and risk of bias was performed using the methodological index for non-randomized studies (MINORS) criteria . MINORS event) and denominator , if possible. Depending on the distribution of proportions, either no or logistic transformation was applied [p value of < 0.05 in the Cochrane Q test and I2 > 50%) was observed, a random-effects model (DerSimonian and Laird) was applied. The predominantly small sample sizes and retrospective study design prevented multivariable meta-regression due to inability to properly adjust for the effects of confounders. As it has been shown to be problematic in meta-analyses of proportions, assessment of publication bias using funnel plots was not performed [If sufficient data were available for evaluation, a meta-analysis of proportions was conducted to calculate the weighted summary overall proportion. These pooled rates represent the average from multiple studies weighted by the inverse of their sampling variances and are presented along with 95% confidence intervals (95% CI) calculated using a normal approximation . The pro applied . When sierformed . All anaOverall, we identified 73 studies comprising 9110 patients who underwent primary BST with curative intent according to our inclusion criteria . The proportion of early and late SV-RC was equally balanced (55.7% vs. 44.3%). Pooled rate of incontinent urinary diversion was 91.1% for all reported cases . In the series of Chahal et al. which compared the outcome of SV-RC and primary RC, 8.3% in the primary RC group had a continent urinary diversion vs. only 3.5% in the SV-RC group. The pooled rate of 5- and 10-year DFS after SV-RC was 54.3%  and 45.6% , respectively.The pooled rate of all subsequently necessary SV-RC was 15.5%  and major gastrointestinal complications  in SV-RC vs. primary RC patients [Only three studies provided a thorough report of complications after SV-RC, while two further studies provided partial reports. Of these, four studies reported the overall complication rate, which ranged between 67 and 72.2% –30. Two patients . Similarpatients .30-day mortality rates for SV-RC-treated patients were generally low (Supplementary Table 2), but were higher than those for primary RC  . CompareI2 = 92%), this increased to 32.9% for studies with longer than 5 years follow-up , while pooled rate for non-response (stable or progressive disease) was 15.5% . Several studies reported that a significant proportion of patients were not salvageable after BST treatment failure (12–52%) [No study focused on analyzing variables that could predict the need for SV-RC. Less than 2% of the patients underwent SV-RC due to non-oncologic reasons , 31, 32.(12–52%) , 33, 34.I2 = 99%). Patients undergoing SV-RC had a higher proportion of cT3 and cT4 diseases compared to those who did not need a SV-RC  [Two large series noted that in patients with an incomplete primary TURBT before BST, the rate of SV-RC was higher compared to the rest of the cohort (e.g. 42% vs. 22%) , 35. In In terms of methodologic quality, MINORS scores ranged from 7 to 16 . Overall, 80.8% (59/73) of all studies included into the meta-analysis were, therefore, found to be without a risk of bias.The prerequisite criteria for BST to be considered as an adequate alternative to primary RC in patients with MIBC need to be addressed. It is obvious that the equivalence of BST to primary RC, the standard of care in this disease space, can only be assessed in a prospective randomized controlled clinical trial. Since such a study is nearly impossible, due to the patients and physicians’ lack of enthusiasm, we have attempted to investigate the criteria that would allow BST to be accepted as an alternative to primary RC . We found that one in five patients treated with primary BST with a curative intent subsequently required SV-RC. However, these patients may have represented only a proportion of failures, as not all patients will be eligible for SV-RC. Failure of BST and subsequent time delay might deprive patients of other treatments alternatives , 33, 34.Despite the significant rate of failure in BST leading to subsequent SV-RC, only 3 out of 73 studies thoroughly reported the outcomes of SV-RC. Even with advances in intra- and perioperative care, SV-RC remains a challenging surgery. Indeed, it leads to overall complication rates ranging between 67 and 72% –30. If sPredictors of SV-RC need to be investigated to improve patient selection. In agreement with the literature on oncologic outcome after BST, our findings suggest that patients with multifocal tumors, hydronephrosis non-organ confined disease, incomplete TURBT or carcinoma in situ should not be selected for BST . DespiteThe main limitation of this study is its retrospective design. Due to the differences in staging techniques, tumor subtypes, inclusion criteria and treatment modalities the final cohort analyzed was found to be very heterogeneous. Also, we identified only three studies that thoroughly reported the outcomes after SV-RC, as most studies only briefly mentioned the rates of necessary SV-RC. Despite an acceptable quality of the studies included, prospective trials assessing precisely the risk of SV-RC and the oncologic outcomes are highly needed.Approximately one-fifth to one-third of patients treated with BST with a curative intent eventually a SV-RC. This procedure has a proportionally high rate of complications and, while primarily entailing a non-tissue sparing (i.e. nerve-sparing) approach, usually is accompanied by an incontinent urinary diversion. The resulting decreased quality of life needs to be taken into consideration during the counselling of MIBC patients regarding the risks, benefits and alternatives (i.e. BST) to primary RC.Supplementary file1 (DOCX 10161 kb)Supplementary file2 (DOCX 36 kb)Supplementary file3 (DOCX 13 kb)Below is the link to the electronic supplementary material.


Severe community-acquired pneumonia (SCAP) is a common critical disease in the intensive care unit (ICU). This study aims to evaluate the clinical significance of miR-181b in SCAP, which has been revealed to be dysregulated in acute respiratory distress syndrome events due to SCAP. There were 50 SCAP patients and 26 healthy volunteers were recruited in this study. The expression of miR-181b was detected by RT-qPCR and the difference between SCAP and healthy controls was evaluated. The diagnosis and prognosis value of miR-181b was assessed by the receiver operating characteristics (ROC), Kaplan-Meier, and Cox regression analysis. miR-181b was significantly downregulated in SCAP compared with healthy controls. The downregulation of miR-181b showed a significant association with the white blood cell count, absolute neutrophils, and the C-reactive protein of patients. The downregulation of miR-181b could distinguish SCAP patients from healthy controls and predicate the poor prognosis of SCAP patients. Downregulated miR-181b serves as a diagnosis and prognosis biomarker for SCAP, which may be useful biological information for the early detection and risk estimation of SCAP. Pneumonia is one of the lower respiratory tract infections, which accounts for the high mortality of patients . Severe Mycoplasma pneumoniae, and via targeting B7-H3, miR-29c exerted inflammatory immune response to M. pneumoniae infection (MicroRNAs (miRNAs) are highly conserved composed of 18-25 nucleotides, which have been demonstrated to play vital roles in the posttranscriptional regulation of gene expression in the pathogenesis of lung disease and infections . Previounfection . miR-181nfection . It was The purpose of this study is to estimate the clinical significance of miR-181 in SCAP and confirm whether miR-181 could be used as diagnosis and prognosis biomarkers for the discrimination of SCAP and the prediction of SCAP development.A total of 50 SCAP patients were enrolled from the ICU of Shanxian Dongda Hospital during 2017-2019. Another 26 healthy volunteers that had normal physical examinations were recruited as the control group. The diagnosis of SCAP patients was based on the presence of pulmonary infiltrates on the chest and the clinical presentation, including cough, sputum production, dyspnea, fever > 37.8 °C. The severity was evaluated by the pneumonia severity index (PSI). Pneumonia developing during hospitalization was excluded. This study was approved by the Ethics Committee of Shanxian Dongda Hospital (No. 2016045) and informed consent was obtained from each participant or their guardians. A 28-day follow-up survey was conducted to obtain the survival information of all participants. Serum samples were collected within 24 h of ICU admission and stored at -80 °C for further analysis.The serum concentration of C-reactive protein was measured by the turbidimetric inhibition immune assay. The lactate dehydrogenase was determined according to the previous study . The abs−ΔΔCt method was used to calculate the relative expression level of miR-181b with U6 as the internal standard. The primer sequences used were as follows: miR-181b forward 5’-GCGGATCATTCATTGCTGTCG-3’, reverse 5’-GTGCAGGGTCCGAGGT-3’; U6 forward 5’-GACCTCTATGCCCAACACAGT-3’, reverse 5’-AGTACTTGCGCTCAGGAGGA-3’.Total RNA was extracted with TRIzol reagent . Extracted RNA was transcribed reversely to cDNA was generated by the PrimeScript RT reagent Kit . The expression of miR-181b was analyzed by the Applied Biosystems 7900 Real-Time PCR system  with the SYBR Green I Master Mix Kit . The 22 test and Pearson’s correlation analysis. Kaplan-Meier analysis and Cox regression analysis were employed to estimate the prognostic value of miR-181b in SCAP. The diagnostic value of miR-181b was evaluated by the receiver operating characteristic (ROC) and the values of area under the curve (AUC) with 95% confidence interval (95% CI) were also calculated. It was statistically significant when P < 0.05.Data were represented as mean ± SD. and analyzed by SPSS version 23.0 software  and GraphPad Prism 7.0 software . Differences between groups were assessed by student’s t-test or one-way ANOVA. The association between miR-181b expression level and the clinical characteristics of patients was estimated by the χP > 0.05). While, the white blood cell count (P = 0.003), absolute neutrophils (P < 0.001), and the concentration of C-reactive protein (P < 0.001) of SCAP patients were significantly higher than those of healthy volunteers.As shown in P < 0.001, P = 0.018), absolute neutrophils (P = 0.035), the concentration of C-reactive protein (P = 0.040), and the PSI scores (P = 0.021) of patients , absolute neutrophils , c-reactive protein concentration , and the PSI scores  were also validated by the Pearson’s correlation analysis  curve. The ROC curve showed that miR-181b could discriminate SCAP patients from healthy volunteers with the area under the curve of 0.883, and the sensitivity and specificity of 0.780 and 0.923, respectively .P = 0.034). Additionally, the results of Cox regression analysis showed that miR-181b  and the PSI scores  served as independent indicators for the prognosis of SCAP patients , which always developed from mild pneumonia with a high incidence rate . NowadayThe major finding of the present study revealed that miR-181b was downregulated in SCAP compared with healthy controls, and showed significant association with the white blood cell count, absolute neutrophils, C-reactive protein concentration, and PSI scores of SCAP patients, which are important clinical and laboratory characteristics of patients . miR-181Previously, miR-181b was identified as biomarkers in a variety of diseases. In colorectal cancer and acute myeloid leukemia, miR-181 could predict the poor survival of patients . miR-181There are several minor limitations to this study. Due to the limitation of recruited patients, the sample size of this study was small and only a severe population was included. Likely, the results of the present study may not be generalizable to a non-selective population of patients with pneumonia. In previous studies that focused on the function of miR-181b in other human diseases, TGF-β was identified as a direct target of miR-181b . FurtherTaken together, miR-181b was downregulated in SCAP and associated with the white blood cell count, absolute neutrophils, C-reactive protein concentration, and the PSI scores of SCAP patients. The downregulation of miR-181b could serve as diagnosis and prognosis biomarkers for the early screening and outcome prediction of SCAP patients.


P ≤ 0.05 were revealed, and 708 of them were up-regulated. Based on the count score, the top within GOs was GO cellular components “extracellular exosome”, and the top KEGG pathway was the metabolic pathway. Ten selected DEGs, i.e. hydroxysteroid (17β) dehydrogenase 8, cyclooxygenase 2, prostaglandin F receptor, progesterone receptor membrane component 1, progesterone receptor membrane component 2, annexin A2, homeobox A10, S-phase cyclin A-associated protein in the ER, SRC proto-oncogene, non-receptor tyrosine kinase, and proliferating cell nuclear antigen were conducted through qPCR to validate microarray data. In conclusion, dietary restriction during the peri-conceptional period causes alterations in the expression of genes encoding proteins involved i.a. in the endocrine activity of the myometrium, embryo-maternal interactions, and mechanisms regulating cell cycle and proliferation.This study hypothesized that female peri-conceptional undernutrition evokes transcriptomic alterations in the pig myometrium during the peri-implantation period. Myometrium was collected on days 15–16 of pregnancy from pigs fed a normal- (n = 4) or restricted-diet (n = 4) from conception until day 9th of pregnancy, and the transcriptomic profiles of the tissue were compared using Porcine (V2) Expression Microarrays 4 × 44 K. In restricted diet-fed pigs, 1021 differentially expressed genes (DEGs) with fold change ≥ 1.5,  Females fed an unbalanced diet exhibit endocrine disruptions, the decreased rate of ovulation, the deteriorated oocytes quality, and the lowered potential of embryos to develop12. Poor nutrition in pregnant females is one of the risk factors of high priority that may evoke fetal growth retardation14. Moreover, an unbalanced diet during pregnancy can cause low birth weight, and excessive growth, obesity, circulatory system disorders, and hypertension in adulthood12. However, the origin and the molecular background of these effects still appear not fully understood.The proper nutrition of a female during the peri-conceptional period impacts the reproductive success, pregnancy outcome, and health of future offspring16. It is believed that alterations in this milieu may be especially hazardous while appearing during the peri-conceptional period20. The composition of the intrauterine environment depends on i.a. maternal diet16, but also secretory activity of uterine tissues, including the myometrium26. Studies performed on pigs demonstrated that the peri-conceptional undernutrition leads to the decrease of the total content of estradiol-17β (E2) in the uterine flushings during the peri-implantation period17. This effect can be a result of the altered steroidogenic activity of the uterus. Furthermore, the previous studies have shown that the peri-conceptional undernutrition induces global transcriptomic changes in conceptuses and the endometrium of females during the peri-implantation period27 suggesting that dietary restriction may affect a variety of mechanisms occurring in the uterus for proper embryo implantation and future development. Until now the effect of dietary restriction on myometrial transcriptome was not studied. However, there was documented an effect of restricted diet on the myometrial expression of methyltransferases, determining the potential for DNA methylation17. Therefore, it cannot be excluded that changes in the myometrial potential for DNA methylation, as an effect of an unbalanced diet, may induce transcriptomic alterations in the tissue, determining its potential and activity.According to the Developmental Origins of Health and Disease concept, the changes in embryos and offspring starting at the early stages of development may depend on the intrauterine milieu18, conceptuses27 of pigs fed a restricted diet during the periconceptional period. This study provides new data concerning molecular alterations in the uterine tissues and conceptuses during the peri-implantation period that occur as the consequence of female peri-conceptional undernutrition.This study hypothesized that undernutrition in females during the peri-conceptional period evokes alterations in the transcriptomic profile of the myometrium during the peri-implantation period. Thus, the current study aimed to determine and compare the transcriptomic profiles of the peri-implantation myometrium collected from females fed a normal- or restricted-diet during the peri-conceptional period. The study was performed on pig model, however, it is intended to imitate deprivation of nutritional habits in women who, during early stages of pregnancy, i.e. during the peri-conceptional period, are not aware of carrying a child. In this study, a particular focus was put on differentially expressed genes (DEGs) that are known to encode proteins crucial for the creation of the proper intrauterine environment, embryo-maternal interactions, and myometrial morphogenesis. Furthermore, there was performed transcription factor enrichment analysis to provide insight into the mechanisms of alterations in the transcriptomic activity of the myometrium in response to dietary restriction. Finally, there were evaluated common DEGs in the myometrium, endometriumP ≤ 0.05) in the myometrium collected during the peri-implantation period from restricted-diet-fed pigs compared to the myometrium collected from normal-diet-fed pigs. Among evaluated DEGs, 69.34% (708 DEGs) were up-regulated and 30.66% (313 DEGs) were down-regulated. The full list of DEGs is presented in Supplementary Table https://www.genecards.org/), three groups of DEGs were evaluated. The first grouped genes which products are involved in the creation of a proper intrauterine environment, i.e.: synthesis of cholesterol, steroid hormones, and prostaglandins in the myometrium, and myometrial response to their action (Group 1: The endocrine activity), second grouped genes which products are involved in the regulation of embryo-maternal interactions during the peri-implantation period and involved in processes associated with the implantation , and third one grouped genes involved in the regulation of myometrial cell proliferation and differentiation, and tissue morphogenesis (Group 3: Mechanisms of proliferation and differentiation). The selected genes within Groups 1, 2, and 3 are presented in Table The transcriptomic analysis evaluated 1021 DEGs  possessed identified DAVID ID , i.a. HMGCR, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, COX2 encoding cyclooxygenase 2, NSDHL encoding NAD(P) dependent steroid-dehydrogenase-like, HSD11B1 encoding hydroxysteroid (11β) dehydrogenase 1, and HSD17B8 encoding hydroxysteroid (17β) dehydrogenase 8.Using a DAVID functional annotations analysis, evaluated up-, and down-regulated DEGs were classified into 50 Kyoto Encyclopedia of Genes and Genomes (KEGG)Genes interaction networks were created for groups of DEGs indicated in Table M) and Real-time PCR (FCRT) are presented in Fig. M values and FCRT values was 0.947 (P ≤ 0.05), confirming that results obtained using microarray are validated.The FC values obtained using microarray analysis , whereas in the endometrium above half of them (58%) were down-regulated27. Thus, the transcriptomic activity in the myometrium and conceptuses during the peri-implantation period in response to dietary restriction during the peri-conceptional period may be mostly up-regulated, while in the endometrium it is rather down-regulated.The present study demonstrated that the short-lasting undernutrition in pigs during the peri-conceptional period alters the transcriptomic profile of the myometrium during the peri-implantation period. Specifically, as a consequence of the females’ nutritional restriction, the expression of 1021 genes encoding proteins with known biological functions has been changed ≥ 1.5-fold, and the majority of them, i.e. 69.34%, were up-regulated. Based on the results of TF enrichment analysis, there are 333 TFs that may be involved in the regulation of the expression of DEGs identified in the current microarray experiment. Interestingly, 24 of these TFs were differentially expressed in response to dietary restriction. Thus, the restriction diet used during the peri-conceptional period may alter the transcriptional activity of the myometrium by affecting the expression of TFs. In the current study, the highest number of gene count was within GO CC. Similar findings were demonstrated previously in the endometrium of restricted-diet-fed pigs31. Until reaching the blastocyst stage, which occurs approximately at day 9 in this species, the embryonic genome undergoes active and passive demethylation31. In consequence of the disruptions in genome-wide methylation and demethylation, embryos are likely to develop with less than fully penetrant phenotypes31. It is known that both restricted and excessive maternal dietary protein intake during pregnancy alter the offspring's epigenetic marks and influence the expression of genes32. Moreover, DNA demethylation mechanisms act during early embryonic development, and a drastic reduction of methylation levels is expected to be found at later preimplantation stages31. Providing that DNA methylation is one of the mechanisms regulating the transcriptomic activity, the selection of a peri-conceptional period for dietary restriction for further transcriptome profiling during later stages of pregnancy is justified. We believe that the myometrium possesses “epigenetic sensitivity”, mainly during intensive changes occurring during early pregnancy, and the epigenetic alterations, including DNA methylation, are one of possible mechanisms underlying the diet-related alterations in the transcriptomic activity of the uterus.In this study, we have selected day 9 as the cut-off of under-nutrition protocol based on epigenetic events, namely methylation profile establishment, occurring during the early stages of pregnancy35. These alterations contribute to the creation of a specific, intrauterine milieu, which is critical for proper embryo-maternal dialog during early pregnancy37. It is worth highlighting that our previous study showed that restricted-diet fed gilts exhibit a decreased intrauterine concentration of E217. It is known that in pigs E2 is one of maternal recognition of pregnancy signal38. Thus, the decrease in its concentration may deteriorate the developmental potential of embryos and impact mechanisms of pregnancy establishment. Notably, E2 is also an important regulator of transcriptional activity of the uterus39. Thus, the alterations in the intrauterine concentration of E2 evoked by dietary restriction17 could stand for an additional possible mechanism underlying the observed diet-related alterations in the transcriptional activity observed in the current and previous study18.The previous studies determined that the myometrium is an endocrine tissue, and its secretory activity differs regarding the physiological status of the female (pregnant vs. estrous cyclic pigs) and in the following days of pregnancy and the estrous cycleHMGCR, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) classified to some of evaluated KEGG pathways30, i.a. metabolic pathways and also to some GOs, i.e. GO BP: oxidation–reduction process (GO:0055114), GO CC: endoplasmic reticulum membrane (GO:0005789), and GO CC: peroxisomal membrane (GO:0005778). The HMGCR is the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, which are substrates required for post-translational modifications of proteins involved in a signaling pathway that regulates embryonic development , and for steroid hormones biosynthesis pathway41. Respecting that porcine myometrium possesses an active steroidogenic pathway34, the observed increased expression of HMGCR in the myometrium of the restricted diet-fed pigs may lead to a higher supply of cholesterol, for further synthesis of steroid hormones in the myometrium. However, the previous studies demonstrated that the dietary restriction during the peri-conceptional period results in the decrease of the total content of E2 in the uterine flushings during the peri-implantation period17. This finding might be underlaid by previously determined diet-related up-regulation of endometrial HSD17B418, encoding hydroxysteroid (17β) dehydrogenase type 4 (17βHSD4), which converts estradiol-17β (E2) to estrone (E1)42. Based on the current results, the expression of HSD17B4 was not altered in the myometrium of restricted-diet-fed pigs, but there was observed an up-regulation of the HSD17B8 gene. This gene codes for 17βHSD type 8 catalyzing the interconversion of androstenedione (A4) to testosterone (T), and E1 to E2. Thus, the diet-related decrease in the uterine content of E2 is dependent rather on endometrial than myometrial steroidogenic activity.Among the evaluated DEGs in the myometrium of restricted-diet-fed pigs, there was HMGCR is co-expressed and co-localized with HSD11B1, coding for 11β-hydroxysteroid dehydrogenase type 1 (11βHSD). The 11βHSD participates in the conversion of cortisone to cortisol44. Therefore, the observed in the current study up-regulation of HSD11B1 in the myometrium indicated the potential of the tissue to produce cortisol. Interestingly, it was previously found that cortisol may decrease the release of luteolytic prostaglandin F2α (PGF2α) from the endometrium45. The expression of cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), and prostaglandin F receptor (PTGFR) were previously demonstrated in the myometrium as well as the active PGF2α release by this tissue in vitro46. The current study has shown that dietary restriction leads to the decreased expression of COX2 and PTGFR in the myometrium. Thus, the action of PGF2α in the myometrium of restricted-diet-fed pigs may be disturbed. The peri-implantation period is accompanied by a molecular dialog between conceptuses and the uterus. This phenomenon can be considered as a semi-inflammatory process47, and PGF2α is recognized as a proinflammatory and immunostimulatory factor48. Moreover, PGF2α regulates the expression of genes potentially important for embryo-maternal interactions49. Disturbances in the potential of PGF2α synthesis and its action in the myometrium of restricted-diet-fed pigs expressed as the decreased COX2 and PTGFR expression may affect the PGF2α-mediated process occurring during the peri-implantation period, which can be detrimental for pregnancy success.Interestingly, 450. Interestingly, the current study documented that the restricted diet resulted in the alterations in the expression of progesterone membrane components, i.e. PGRMC1 (up-regulated) and PGRMC2 (down-regulated). Furthermore, PGRMC1 mediates P4-dependent control of the cell cycle, proliferation, and apoptosis, whereas PGRMC2 was found to inhibit cell migration53. Notably, the decreased expression of PGRMC2 was detected also in porcine conceptuses collected from restricted-diet-fed pigs27. The alterations of PGRMCs expression in response to the dietary restriction may affect the non-genomic action of P4, and impact cell cycle, proliferation, and apoptosis of myometrial cells. Uterine development and morphogenesis during early pregnancy are also regulated by homeobox A family proteins (HOXA)54. Previous studies demonstrated that the expression of HOXA10 and HOXA13 is increased in the myometrium of early pregnant pigs46. Thus, the decreased expression of HOXA3 and HOXA10 in the myometrium of restricted diet-fed pigs may affect cell cycle, myocyte proliferative capacity and differentiation, and development of the myometrium required for proper development of the uterus during pregnancy.The proper embryo-maternal interactions require signals triggered by Panaphase-promoting complex subunits, i.a. ANAPC5, ANAPC7, ANAPC16, and the expression of proliferating cell nuclear antigen (PCNA) and SRC proto-oncogene, non-receptor tyrosine kinase (SRC) was significantly increased, while the expression of S-phase cyclin A-associated protein in the endoplasmic reticulum (SCAPER) was decreased. The genes encoding ANAPC is classified to the GO CC term “anaphase-promoting complex” (GO:0005680). The ANAPC regulates the proper chromosome segregation during mitosis, mitotic exit, and the maintenance of the G1 phase of the cell cycle55. PCNA may be considered as a regulator of DNA replication, while SRC is involved in the regulation of proliferation, differentiation, and cellular viability57. It was found that the decreased content of SCAPER contributes to the decreased level of cytoplasmic cyclin A resulting in the delay of G1/S phase transition during the cell cycle58. Thus, the dietary restriction in females during the peri-conceptional period may determine the myometrial cells' fate during the peri-implantation period.Notably, the restricted diet during the peri-conceptional period evokes the alteration in the several genes encoding proteins involved in the regulation of the cell cycle, proliferation, and differentiation. This study evaluated that the expression of genes encoding ANXA2 (annexin 2) in the myometrium of pigs during the peri-implantation period. The ANXA2 is classified to many GO terms among GO CC, i.a. extracellular exosome (GO:0070062), cytosol (GO:0005829), extracellular matrix (GO:0031012), and membrane raft (GO:0045121), and among GO MF, poly(A) RNA binding (GO:0003723). In general, annexins are a family of proteins involved in many cellular functions, including vesiculation and cell membrane repair, and ANXA2 induces membrane folding and blebbing initiated from membrane structural defects59. The previous studies demonstrated that myometrial expression of ANXA2 was greater in pigs during the peri-implantation period than during luteolysis46. In the current study, the abundance of the ANXA2 mRNA transcript was found to be greater in the myometrium of the restricted-diet-fed pigs. Further bioinformatical analysis revealed that ANXA2 is co-expressed with genes encoding other annexins (ANXA1 and ANXA11), intercellular adhesion molecules , and platelet/endothelial cell adhesion molecule 1 (PECAM1), which exhibited up-regulated expression in the myometrium of restricted-diet-fed gilts. Interestingly, these genes encode for proteins involved in processes of embryo apposition and adhesion to the uterine wall as well in the induction of angiogenesis60. Thus, the mild nutritional restriction during the peri-conceptional period may affect the mechanisms determining the myometrial integrity and angiogenesis during the peri-implantation period.The current study demonstrated that the restricted diet during the peri-conceptional period also leads to the up-regulation of 18, and conceptuses27 collected from restricted-diet-fed pigs. Among DEGs which are common for uterine tissues and conceptuses there is MCL1 apoptosis regulator, BCL2 family member (MCL1), which expression in restricted diet-fed pigs was increased in the myometrium (the current study) while decreased in the endometrium and embryos27. The MCL1 is considered an anti-apoptotic protein, and its down-regulation leads to the activation of caspases involved in apoptosis61. Thus, the interesting phenomenon of increased gene expression in the myometrium and decreased gene expression in the endometrium and embryos in response to the restricted diet may affect the rate of apoptosis during the peri-implantation period. Furthermore, in the myometrium (the current study), the endometrium18, and embryos27 of the restricted diet-fed pigs there was observed the up-regulation of ACP5, which is involved in the transport of iron from the endometrium to embryos62. The ACP5 is classified to the “extracellular exosome” (GO:0070062) GO CC term. This finding indicates that the restricted diet may potentially affect the uterine and conceptuses' capacity to produce the extracellular vesicles that may participate in the embryo-maternal cross-talk63. It is worth highlighting that both ACP5 and FTH1, which is the gene encoding ferritin heavy chain 1, were up-regulated in the myometrium (the current study), the endometrium18 and conceptuses27 of restricted diet-fed pigs. Interestingly, FTH1 is a major intracellular iron storage protein that may function in the protection from oxidative stress64. Thus, the up-regulation of FTH1 and ACP5 genes in uterine tissues and conceptuses may lead to the altered distribution of iron in embryo-uterine interspace. Thus, the described alterations in the expression of ACP5, FTH1, GSTA4 and GPX3, occurring in the uterine tissues and conceptuses of restricted-diet-fed pigs, may also be considered as mechanisms of intrauterine protection from oxidative stress.This study assessed the transcriptomic alterations in the myometrium (the current study), endometriumKLF4, encoding for Krüppel-like factor 4. KLF4 is involved in the regulation of numerous biological processes including cell development and cell division, apoptosis, and cell programming, and its down-regulation is often coupled with tumor progression65. This study showed that nutritional restriction increases the transcriptional activity of KLF4 in the myometrium. Importantly, the previous studies showed that an up-regulation of KLF4 induces cell cycle arrest65. Having in mind that this study evaluated diet-related alterations in the transcriptional activity of genes coding for proteins involved in the regulation of cell cycle and determining the proliferative potential of the myometrium, i.a. PGRMC genes, HOXA genes, ANAPC genes, SRC, SCAPER, PCNA, and these changes may be connected with the altered expression of KLF4. Among differentially expressed TFs there was also RELA coding for RELA Proto-Oncogene, NF-KB Subunit. RELA is a part of the nuclear factor kappaB (NF-kappaB) family, regulating the expression of i.a. pro-inflammatory mediators. The expression of RELA was increased in the myometrium in response to dietary restriction. It is worth highlighting that pregnancy might be recognized as a semi-inflammatory process where the action of cytokines, tumor necrosis factor (TNF), and COX2 is crucial for proper implantation47. In this study, we observed diet-related down-regulation in COX2 expression, and this alteration might be connected with the altered expression of RELA. However, the applied undernutrition protocol did not contribute to alterations in the expression of pro-inflammatory cytokines and TNF what suggests that there must appear additional mechanism controlling the transcriptional activity of the myometrium of restricted-diet-fed pigs. One may not exclude that this mechanism relies on epigenetic mechanisms, including DNA methylation, but this assumption needs further investigation.For a better understanding of the mechanisms underlying changes in the transcriptional activity of the myometrium occurring in response to a restricted diet, we have identified TFs casually responsible for changes in the expression of the evaluated DEGs. Indication of TFs which may be involved in the regulation of the transcriptional activity of the evaluated DEGs is promising, but still challenging and show that applied undernutrition protocol may evoke a broad spectrum of transcriptomic alterations. Notably, the peri-conceptional undernutrition caused alterations in the transcriptional activity of 24 of the evaluated TFs. Among these differentially expressed TFs, there is In conclusion, the observed in the current study alternations in the transcriptomic profile of the myometrium show that the short-lasting 30% decrease of food intake by pregnant females during the unique peri-conceptional period may disturb the proper course of biological processes occurring in the myometrium during the peri-implantation period. Among the observed alterations, increased potential for the synthesis of steroid hormones, decreased potential for prostaglandins synthesis and action, decreased proliferative capacity, and increased adhesion among myocytes were determined. The observed transcriptomic alterations of the myometrium might jeopardize proper implantation and thus, pregnancy success, but this requires further in vivo studies. Moreover, the results documented that the transcriptomes of the myometrium, endometrium, and conceptuses during the peri-implantation period respond differently to female dietary restriction during the peri-conceptional period.66, and all methods were carried out following relevant guidelines and regulations indicated where appropriate.All experiments were approved by the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland—decision No. 54/2015/DTN. The study was carried out in compliance with the ARRIVE guidelinesSus scrofa Domestica L., Polish Landrace × Great White Polish, weighing 95 to 110 kg) were naturally bred on the first and second day of estrus of their second estrous cycle. Gilts assigned to the control group (n = 4) were fed a normal diet, i.e. daily 3 kg/gilt of forage since the first sign of the estrus, during natural mating, and until slaughter. Gilts assigned to the experimental group (n = 4) were fed a restricted diet, i.e. daily 2.1 kg/gilt of forage, which is a 30% food restriction, during the peri-conceptional period , and next fed a normal diet until slaughter.The post-pubertal pigs  and 100 IU mL−1 of streptomycin , and transported to the laboratory. The day of pregnancy was confirmed by the morphology of ovaries and conceptuses flushed from uterine horns as well as the concentration of P4 in peripheral blood71. Next, uterine horns were opened longitudinally, the perimetrium tissue was discarded by careful scraping, and the myometrial cross-sections were collected with scissors and snap-frozen in liquid nitrogen (− 196 °C). Samples were stored at – 80 °C until further analysis.Gilts from both groups were slaughtered in a local abattoir  during the peri-implantation period, specifically on day 16 after the first mating. Respecting the mating protocol and effectiveness of mating, the animals were on day 15 to 16 of pregnancy. Immediately after slaughter uteri were collected and placed into ice-cold phosphate-buffered saline (PBS) containing 100 IU mLg, for 10 min at 4 °C and the supernatant was transferred to a 350 µL of propane-2-ol  beforehand chilled at − 20 °C. Next, samples were mixed well by vortexing and incubated for 15 min on ice assuring brief mixing every 3 min. Next, samples were centrifuged 12,000×g, for 10 min at 4 °C, and the supernatant was discarded. RNA pellets were washed twice with 1 mL of 75% ethanol  beforehand chilled at − 20 °C. The RNA pellets were suspended in 30 µL of DEPC-treated water . The initial analysis of concentration and purity of extracted RNA was determined using Tecan with i-control software , and the initial test for RNA integration was performed using gel electrophoresis. Samples with a concentration higher than 100 ng/µL, optical density (OD) from 1.8 to 2.0, and integral electrophoretically were used in the next steps of analysis. The samples that fulfilled these criteria were tested using microcapillary gel electrophoresis in 2100 Bioanalyzer  to determine RNA integrity number (RIN). Samples with RIN ≥ 7.5 were used for microarray analysis.RNA was extracted from 30 mg-weighting myometrial slices using 500 mL of TRI Reagent . Samples were initially homogenized on ice using a homogenizer TissueRuptor . Then, a 150 µL of chloroform  was added, samples were mixed thoroughly and incubated for 10 min at 4 °C. Next, samples were centrifuged 12,000×72. The specific workflow of the experiment was described previously46. Briefly, 400 ng aliquots of RNA were used to synthesize cRNAs samples labeled with CY3 or CY5. Fluorochrome-labeled cRNA was purified on RNeasy Mini Spin columns . After determination of cRNA concentration and labeling efficiency, samples were conducted through the cRNA fragmentation step and mixed with a hybridization buffer. Then, samples were applied on microarrays following the classic class comparison experiment design, using a dye-swap to prevent dye bias74. After hybridization (for 17 h at 65 °C), microarrays were washed, dried, and scanned using Agilent High-Resolution C Microarray Scanner . Fluorescence intensity was calculated using Agilent Feature Extraction software v. 10.5.1 , the outlier spots were filtered, and the background correction and dye normalization (linear and LOWESS) were performed. The obtained data were analyzed using GeneSpring GX 13.0 software . The transcripts with fold change (FC) ≥ 1.5 and P ≤ 0.05  were aligned with the Sus scrofa transcriptome (txid9823) using the BLAST tool for identification of encoded gene name. When more than one differentially expressed transcript was assigned to one gene name, the FC and P-values of these transcripts were averaged and recognized as one differentially expressed gene (DEG). DEGs that were found in both up-regulated and down-regulated gene lists were omitted in further analyses.The microarray analysis was performed using RNA samples isolated from the myometrium collected from restricted-diet-fed pigs  in comparison to the normal-diet-fed pigs  using Porcine (V2) Expression Microarrays 4 × 44 K  according to the Two-Color Microarray-Based Gene Expression Analysis Protocol . Four independent biological replicates per group, i.e. experimental and control group, allowed to obtain an experiment power of 80% and a false discovery rate (FDR) of 0.1v. 6.8 (accession June 2021)76, annotations were limited to Sus scrofa and DAVID IDs of uploaded DEGs were determined. DEGs with determined DAVID ID were classified into gene ontology (GO) terms within biological processes (BP), cellular components (CC), and molecular functions (MF) within GO Direct. DAVID GO IDs were defined using the Quick GO database (http://www.ebi.ac.uk/QuickGO). Then, up- and down-regulated DEGs were uploaded together to DAVID for further visualization of KEGG pathways78. In the analyses, only GO terms and KEGG pathways with FDR-adjusted P-values ≤ 0.05 were considered.The full list of DEGs was first divided into two lists: a list of up-regulated DEGs and down-regulated DEGs. Both lists were uploaded separately to Database for Annotation, Visualization and Integrated Discovery (DAVID) 79, using Homo Sapiens as a reference genome. GO-BP-based weighting was used to determine the interactions within co-expression, co-localization, genetic interaction, and physical interaction categories, and each network was enriched with 10 resultant genes.The list of evaluated DEGs was then used to create groups of DEGs encoding proteins involved in the: 1/The endocrine activity, 2/Embryo-maternal interactions, and 3/Mechanisms of proliferation and differentiation. For these groups, the gene interaction networks were created with GeneMANIA prediction server80. For validation procedure, there were selected five up-regulated and five down-regulated genes expressed in the myometrium of restricted-diet-fed pigs. cDNA samples were amplified with POWER SYBR Green PCR Master Mix in AriaMx apparatus with AriaMX Software v. 1.3 . When available, primer sequences were obtained from references , or were designed using BLAST tool provided by NCBI. The reaction parameters were: 1/initial denaturation for 10 min at 95 °C (one cycle), 2/denaturation for 15 s at 95 °C, primer annealing for 1 min, at temperature specific to primers set  following the protocol provided by the manufacturer and rules described by Bustin et al.RT).Calculation of fold change (FC) values for data obtained with Real-Time PCR  and FC values obtained after the microarray experiment (FCM) were used to calculate multiple regression coefficients (R)84.The FC values obtained after Real-Time PCR (FChttps://bio.tools/TFEA.ChIP)85, and a GSEA-like analysis method. There were selected default options: source organism: human, plot type: Enrichment Scores ChiP-Seq source database: ReMap2020 & GeneHancer Double Elite. The output list of TFs was screened for duplicates, which were removed from further analysis. Next, the VENN diagram was created using lists of the TFs and DEGs to evaluate TFs that were differentially expressed (DE) in the myometrium of restricted-diet-fed pigs.Transcription factor (TF) enrichment analysis was performed using human TF dataset with rank test Kolmogorov–Smirnov rank test, and the TFEA.CHiP tool , endometriumSupplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.